The Alphavirus genus in the Togaviridae family contains over 30 members, many of which represent an unquestionable, but often underappreciated, public health threat. In spite of the ability of alphaviruses to cause broad epidemics and severe human diseases, no efficient vaccines or therapeutic means have been developed against any alphavirus infections. This is primarily a result of our insufficient knowledge of their biology, mechanism of replication and interaction with the host. Alphavirus-host cell interaction is particularly poorly understood. In our recent studies, we have made new steps towards understanding functions of one of four alphavirus nonstructural proteins, nsP3. This protein has unique structural characteristics and so far, no functions have yet been assigned to it. Our preliminary data demonstrate that alphavirus nsP3 exhibits both common and virus- and host-specific functions in adaptation of cellular environment for efficient virus replication. These functions are mediated by its carboxy terminal, hypervariable domain, which is intrinsically disordered and thus, can interact with a wide range of host factors. In the research covered by this proposal, we will use a combination of biochemical, molecular and virological methods to characterize cellular proteins interacting primarily with the nsP3 of encephalitogenic Venezuelan equine encephalitis and eastern equine encephalitis viruses. We will perform detailed mechanistic studies of the nsP3-host protein complexes and delineate their functions in virus-specific modifications of the intracellular environment and defining viral pathogenesis. Investigation of the mechanism of the nsP3 functions will have a strong impact on understanding alphavirus replication and will provide new targets for development of antiviral therapy and new approaches for rational design of alphavirus vaccines candidates.